Chapter-10---Enzymatic-Browning_2013_Biochemistry-of-Foods

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Biochemistry of Foods. DOI: http://dx.doi.org/10.1016/B978-0-12-242352-9.00010-2
Copyright � 2013 Elsevier Inc. All rights reserved.
Chapter 10
387
Enzymatic Browning
Vera Lúcia Valente Mesquita and Christiane Queiroz
Departamento de Nutrição Básica e Experimental, Instituto de Nutrição, Josué de Castro, University of Rio de Janeiro, Brazil
Chapter Outline
I. Introduction 387
A. Historical Aspects of Polyphenol
Oxidase 3
87
II. Characteristics of Polyphenol Oxidase 388
A. Structure and Sequence 3
88
B. Mechanism of Reaction 3
89
C. Biological Significance of Polyphenol
Oxidase in Plants 3
91
D. Phenolic Compounds in Food Material 3
93
1. Simple Phenols 3
94
2. Phenolic Acids 3
94
3. Cinnamic Acid Derivatives 3
94
4. Flavonoids 3
95
E. Laccase 3
97
F. Specificity of Polyphenol Oxidase 3
98
III. Polyphenol Oxidase in Foods and Food
Processing 400
A. Role in Tea Fermentation 4
00
B. Shrimps and Crustaceans 4
01
IV. Control or Inhibition of Enzymatic
Browning 403
A. Exclusion of Oxygen 4
03
B. Chemical Inhibitors of Polyphenol
Oxidase 4
04
C. Thermal Processing 4
08
D. High-Pressure Processing 4
09
E. Gamma Irradiation 4
09
F. Pulsed Electric Field 4
10
G. Other Technologies 4
11
References 412
I. INTRODUCTION
Enzymatic browning is a phenomenon which occurs in many fruits and vegetables, such as potatoes, mushrooms,
apples, and bananas. When the tissue is bruised, cut, peeled, diseased, or exposed to any abnormal conditions, it
rapidly darkens on exposure to air as a result of the conversion of phenolic compounds to brown melanins
(Figure 10.1).
The international nomenclature for the enzymes involved in the browning reaction has changed. The first enzyme,
monophenol monoxygenase or tyrosinase (EC 1.14.18.1), initiates the browning reaction, which later involves
diphenol oxidase or catechol oxidase (EC 1.10.3.2) and laccase (EC 1.10.3.1). Catecholase oxidase will be referred
to as ‘polyphenol oxidase’ (PPO) in this chapter. This enzyme requires the presence of both a copper prosthetic
group and oxygen. The copper is believed to be monovalent in the case of the mushroom PPO and divalent in the case
of the potato enzyme (Bendall and Gregory, 1963). PPO is classified as an oxidoreductase, with oxygen functioning
as the hydrogen acceptor. The enzyme is widely distributed in higher plants and fungal and animal tissue and
was reviewed by Swain (1962), Mathew and Parpia (1971), Mayer and Harel (1979), Vamos-Vigyazo (1981),
Mayer (1987), and (2006).
A. Historical Aspects of Polyphenol Oxidase
The earliest work is attributed to Lindet, who in 1895 recognized the enzymatic nature of browning while working on
cider. At the same time Bourquelot and Bertrand commenced studies on mushroom tyrosine oxidase. Subsequently,
Onslow in 1920 showed that enzymatic browning of plant tissue in air was attributed to the presence of o-diphenolic
http://dx.doi.org/10.1016/B978-0-12-242352-9.00010-2
http://dx.doi.org/10.1016/B978-0-12-242352-9.00010-2
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FIGURE 10.1 Browning disorder in ‘Conference’ pears after 4 months in browning-inducing storage conditions (no cooling period, 1% O2,
10% CO2, L1�C) (Reprinted from Franck et al., 2007). � 2007 with permission form Elsevier.
388 Chapter | 10 Enzymatic Browning
compounds, such as catechol, protocatechuic acid, and caffeic acid, plus the appropriate enzymes (oxygenases). The
product of this reaction was thought to be a peroxide which reacted with a ‘chromogen’ to form a brown pigment.
Many fruits and vegetables, including apples, pears, apricots, and potatoes, were all found to be rich in phenolic
compounds and oxygenases. Other fruits, such as citrus, pineapples, and redcurrants, lacked these substances and
were thus termed ‘peroxidase plants’.
This distinction was eliminated when it was shown that peroxidase and catalase were present in both groups of
plants and in fact were ubiquitous in plant tissues. The term ‘oxygenase’ was subsequently replaced by ‘phenolase’ or
‘polyphenol oxidase’. In 1937, Kubowitz demonstrated that PPO was a copper-containing enzyme.
II. CHARACTERISTICS OF POLYPHENOL OXIDASE
A. Structure and Sequence
In order to understand the physiological role of PPO, much research has been conducted to identify gene expression
and protein sequences of the PPO of different plants. It is known that plant PPOs are synthesized as preproteins and
contain putative plastid transit peptides at the N-terminal region, which target the enzyme into chloroplasts and
thykaloid lumen (Marusek et al., 2006). Molecular studies have described PPO as a single genome or as a multiple
gene family. For example, the cherimoya PPO gene was shown to be present in one copy of the genome and had
a very different nucleotide sequence from other published sequences. Despite these divergences, it presented
conserved proteins in the proposed active site and also basic and strategic amino acids related to active site acces-
sibility, protein structure, and stability (Prieto et al., 2007). In Fuji apple plants the expression of two PPO genes
(APO5 and MD-PPO2) was observed during vegetative and reproductive development and in response to wounding.
However, they were not expressed at the same moment or by the same stress stimulus. The maximum level of APO5
messenger RNA (mRNA) was observed in damaged fruits after 24 hours, while the expression of MD-PPO2 was not
increased, suggesting that the selective activation of individual genes under different conditions may reflect the
existence of distinct signal transduction pathways to turn on the different PPO genes (J. Y. Kim et al., 2001). In
addition, seven genes were identified from tomato (Newmann et al., 1993) and five distinct PPO cDNAs were isolated
from potato plants (Hunt et al., 1993).
The existence of multiple genes leads to the presence of PPO isozymes and the variability on protein sequences
observed in the studies explains the differences among enzymes extracted from distinct sources. Two izoenzymes
were present in persimmon, Fuji apple (J. Y. Kim et al., 2001), coffee (Mazzafera and Robinson, 2000), and artichoke
(Aydemir, 2004), while medlar PPO showed four isoforms (Dincer et al., 2002). PPO purified from field bean is
a tetramer with 120 kDa and exists as a single isoform in the seed (Paul and Gowda, 2000), while pineapple PPO has
three isoenzymes, with the major isoform being a tetramer of identical subunits of 25 kDa (Das et al., 1997).
FIGURE 10.2 X-ray structure of polyphenol oxidase (PPO) from Vitis vinifera. (A) Ribbon model showing the overall ellipsoidal shape, two
b-sheets, and the dicopper center within four-helix bundle. (B) Ca representation of V. vinifera PPO (blue) overlapping with those PPO from sweet potato
(yellow) and Streptomyces castaneoglobisporus (green) See online for a colour version of this figure (Virador et al., 2010). Reprinted with permission,�
2010 American Chemical Society.
389II. Characteristics of Polyphenol Oxidase
The molecular mass of PPO from other species has been reported as follows: mulberry, 65 kDa (Arslan et al., 2004);
Henry chestnuts, 69 kDa (Xu et al., 2004); and pulp pine, 90 kDa (Lima et al., 2001).
The crystal structure of PPO has been elicited for sweet potato, grape, and Neurospora crassa. The overall
structure of the PPO from Vitis vinifera is a monomeric protein of 38.4 kDa. It is an ellipsoid with dimensions
56.7� 48.0� 48.3 Å3 (Figure 10.2A, B). The secondary structure is primarily R-helical with the core of the protein
formed by a four-helix bundle composed of R-helices R4, R5, R12, and R14 (Figure 10.2A). Figure 10.2(B) shows
overlapping images of the three existing PPO structures. There are a few areas where the folding is slightly different.
For the most part, these differences occur on the surface and do not involve changes in R-helices or b-strands (Virador
et al., 2010).
B. Mechanism of Reaction
The mechanism of action proposedfor PPO is based on its capacity to oxidize phenolic compounds. When the tissue
is damaged the rupture of plastids, the cellular compartment where PPO is located, leads to the enzyme coming into
contact with the phenolic compounds released by rupture of the vacuole, the main storage organelle of these
compounds (Mayer and Harel, 1979).
PPO catalyzes two types of reaction: cresolase activity, in which monophenols are hydroxylated to o-diphenols,
and catecholase activity. The catecholase or diphenolase type of reaction is best illustrated by the oxidation of
catechol, an o-diphenol which is a commonly used laboratory substrate:
The cresolase or monophenolase activity involves hydroxylation of monophenols to o-diphenols, as shown by the
oxidation of L-tyrosine to 3,4-dihydroxyphenylalanine, which occurs in potatoes (Schwimmer and Burr, 1967):
The active site of PPO consists of two copper atoms coordinated with six histidines, and exists in three oxidation
states: deoxy (Ed), met (Em), and oxy (Eo) (Figure 10.3). One of the Cu2þ atoms is bound to monophenols while
N
N N
N
Cu
+
Cu
+
O
N
N
Cu
+2
Cu
+2
N
N
H
O
N
N
Cu
+2
Cu
+2
N
NO
-
-
(a) (b) (c)
FIGURE 10.3 Polyphenol oxidase structure: (a) deoxyPPO, (b) metPPO, and (c) oxyPPO. (Adapted with permission from Espı́n et al., 1998.) �
2010 American Chemical Society.
EmM EmD
EoD EoM
Ed
+
O2
Q
k7 k5
k2
k-2k1
k-1
k3
k4
k-46k
k -6
k8
k-8
M + E m + D
D + E o + M
SCHEME 10.1 Kinetic reaction mechanism of polyphenol oxidase (PPO) on monophenols and o-diphenols. Em: metPPO; Ed: deoxyPPO; Eo: oxyPPO;
M: monophenol; D: diphenol; Q: o-quinone. (Reprinted with permission, Espı́n et al., 1998). � 1998 American Chemical Society.
390 Chapter | 10 Enzymatic Browning
diphenols bind to both of them. As shown in the reaction mechanisms, monophenolase activity is intimately coupled
to diphenolase activity and produces two electrons. These are required to incorporate one oxygen atom into the
monophenol substrate.
The structural and kinetic mechanisms for the hydroxylation of monophenols (M) and the oxidation of
o-diphenols (D) to o-quinones (Q) catalyzed by PPO have been established (Fenoll et al., 2004; Espı́n et al.,
1998; Rodriguez-Lopes et al., 1992). Monophenolic substrate initially coordinates to an axial position of one
of the coppers of Eo that leads to the hydroxylation of monophenol by the bound peroxide, loss of water and
formation of the enzymeediphenol (EmD) complex. This complex can either render free diphenol or undergo
oxidation of the diphenolate intermediate that bound to the active site, giving a free quinone and a reduced
binuclear copper on the enzyme site (Ed). OxyPPO is then regenerated after the binding of molecular oxygen
to Ed (Rodriguez-Lopez et al., 1992). When a diphenol is present in the medium, this substrate binds to both
Eo and Em to give EoD and EmD intermediates, which, in turn, give rise to two quinones (Orenes-Piñero
et al., 2005) (Scheme 10.1).
The presence of a dead-end complex, EmM, is related to the occurrence of a lag period in the monophenolase
activity of PPO (Rodriguez-Lopez et al., 1992) that can be reversed by the addition of small quantities of reducing
agents or o-diphenols as cosubstrates.
Quinone formation is both enzyme and oxygen dependent. Once this has taken place, the subsequent reactions
occur spontaneously and no longer depend on the presence of PPO or oxygen. Joslyn and Ponting (1951) summarized
those chemical reactions which may account for the formation of brown melanins.
The first reaction is thought to be a secondary hydroxylation of the o-quinone or of excess o-diphenol:
391II. Characteristics of Polyphenol Oxidase
The resultant compound (triphenolic trihydroxybenzene) interacts with o-quinone to form hydroxyquinones:
Hydroxyquinones undergo polymerization and are progressively converted to red and redebrown polymers, and
finally to the brownmelaninswhich appear at the site of plant tissue injury (Matheis andWhitaker, 1984;Whitaker, 1972).
The ratio of diphenolase to monophenolase activity depends on the plant source and can vary from 1:10 to 1:40
(Vamos-Vigyazo, 1981). Sanchez-Ferrer et al. (1988) partially purified PPO from Monastrel grapes and identified
both cresolase and catecholase activity. Rocha and Morais (2001) also found cresolase and catecholase activity in
Jonagorad apple PPO. Cresolase activity was described for strawberry and pear by Espı́n et al. (1997a, b). Several
plants, however, have been found to be devoid of monophenolase activity, including coffee (Mazzafera and Robinson,
2000), broccoli florets (Gawlik-Dziki et al., 2007), butter lettuce (Gawlik-Dziki et al., 2008), and cashew apple
(Queiroz et al., 2011).
C. Biological Significance of Polyphenol Oxidase in Plants
The role of PPO in the living intact cell has until recently remained somewhat obscure. Early studies suggested its
involvement as a terminal oxidase in respiration (James, 1953) or in the biosynthesis of lignin (Mason et al., 1955).
This was later discounted in studies by Nakamura (1967), who examined the role of three enzymes isolated from the
latex of the Japanese lacquer tree (Rhis vermicifera): phenolase; peroxidase; and laccase. Of these enzymes only
peroxidase was involved in lignification. Recent studies have demonstrated the role of laccase in lignification
(Srebotnik and Hammel, 2000; Arora et al., 2002; Shleev et al., 2006). The mechanism is discussed in Section II, E.
Another possible role for PPO is in the biosynthesis of betalain. Red beet (Beta vulgaris) and common portulaca
(Portulaca portiflora) present PPO that hydroxylates tyrosine, forming 3,4-dihydroxylphenylalanine (DOPA), and
oxidizes DOPA to dopaquinone (Steiner et al., 1996, 1999). Enzymatic activity is complemented by dioxygenase
activity, leading to betalain formation. However, more evidence is needed to confirm this function.
PPO is restricted to the plastids. The enzyme appears to be in a latent form and bound to the thylakoid membrane,
where the photochemical reactions of photosynthesis occur. The latent form can be activated in the presence of fatty
acids (Siegenthaler and Vaucher-Boniour, 1971), detergents (Sellés-Marchart et al., 2006), or trypsin (Tolbert, 1973).
Pinto et al. (2008) isolated soluble and insoluble fractions of PPO from cowpea plants and reported activation by
sodium dodecyl sulfate, a detergent, only in the soluble fraction, suggesting that insoluble PPO was purified in the
active form.
In intact cells PPO seems to have little activity towards phenolics, which are located in the vacuole somewhat
isolated from the plastid. Since the enzyme functions normally only when the cells are damaged or undergo
senescence, it may also have a protected role, as proposed originally by Craft and Audia (1962). PPO is involved in
phenolic metabolism when the plastid and vacuole contents are mixed. This occurs during senescence, when the
integrity of the cell is disrupted, and the activation was reported by Goldbeck and Cammarata (1981). However,
recent studies have demonstrated higher activity during development of the plant. Yu et al. (2010) evaluated PPO
activity in longan fruit pericarp collected from 30 days after full bloom to mature fruit. They observed the highest
enzymatic activity at first stage, followed by a sharp decrease, and then a slow increase in the final stage. These data
are also in agreement with those described previously by Yang et al. (2000) and Sun et al. (2009). Ayaz et al. (2008)
investigated PPO activity in medlar fruits during ripening and overripening and showed that, in mature fruit, the
reaction velocity was higher than in overripe fruit. This could indicate that the enzyme was more active than in ripe
and overripe fruits. A molecular study by J. Y. Kim et al. (2001) showed differential expression of two PPO genes in
apple. The authors investigated the mRNA of PPO extracted from flower, fruit, and leaf in different stages and
blotting analysesshowed higher expression in the earlier stages (Figure 10.4).
Enzymatic browning also results following injury to the fruit or vegetable, causing disruption of the plastid,
activation of latent PPO, and catalysis of phenolics released from the vacuole. Activation of PPO by biotic or
abiotic stress had been demonstrated by several studies. Apple PPO showed maximum activity after 24 hours of
APO5
MD-PPO2
APO5 APO5
MD-PPO2 MD-PPO2
S
ta
g
e
 1
S
ta
g
e
 2
S
ta
g
e
 3
S
ta
g
e
 4
S
ta
g
e
 1
S
ta
g
e
 2
S
ta
g
e
 3
S
ta
g
e
 4
1
 c
m
3
 c
m
5
 c
m
Flower Fruit Leaf
(A) (B) (C)
FIGURE 10.4 Tissue-specific expression of two Fuji apple polyphenol oxidase (PPO) mRNAs. Total RNAs were isolated from the different
developmental stages of (A) flower, (B) fruit, and (C) leaf tissues as indicated (Reprinted from J. Y. Kim et al., 2001). � 2001, with permission from
Elsevier.
392 Chapter | 10 Enzymatic Browning
wounding, whereas in cowpea PPO, the activity reached the maximum after 48 hours of wounding (J. Y. Kim et al.,
2001; Pinto et al., 2008). Temperature is an important factor in determining the level of activation since PPO is
thermosensitive. Queiroz et al. (2011) studied the effect of mechanical injury and storage for 24 hours at different
temperatures on cashew apple PPO (Table 10.1). Five-fold higher activation was observed in fruits kept at 2�C and
27�C, but low activity at the higher temperature studied (40�C). Environmental stress can also modulate PPO
activity. Stress situations are related to enhanced activity of phenylalanine ammonia-lyase, which regulates the
synthesis of phenolic compounds, increasing the content of PPO substrates in plant cell (Dixon and Paiva, 1995).
Tegelberg et al. (2008) found higher enzymatic activity in leaves of Betula pendula after exposure to elevated
carbon dioxide (700 ppm) associated with elevated temperature (2.5�C higher than ambient temperature) and
elevated ultraviolet (UV)-B radiation (7.95 kJ/m2/day). On the other hand, Thipyapong et al. (2004b) reported
higher tolerance to water stress in transgenic tomato with suppressed PPO than in non-transformed plants and in
overexpressing PPO. However, in these conditions the authors also observed upregulation of two PPO genes,
probably related to stress resistance. Rivero et al. (2001) submitted watermelon and tomato to cold and heat stress
and observed that PPO activity was decreased and phenylalanine ammonia-lyase activity increased, leading to an
accumulation of polyphenols in stressed plants. These findings demonstrate the different ways that plants use to
defend against an undesirable situation.
The enzyme appears to play an important role in the resistance of plants to infection by viruses, bacteria, and fungi
(Tyagi et al., 2000; Mohammadi and Kazemi, 2002; Wang and Constabel, 2004). Under these conditions the activity
of the enzyme increases with the production of insoluble polymers which serve as a barrier to the spread of infection
in the plant. Alternatively, some of the intermediates in the oxidative polymerization of polyphenols prevent or
reduce infection by inactivating or binding some labile plant enzymes or viruses. Mahanil et al. (2008) observed
increased resistance to the common cutworm [Spodoptera litura (F.)] in transgenic tomato plants overexpressing PPO
(Figure 10.5). The study also showed that increased PPO activity led to higher larval mortality. According to the
authors, the efficiency of conversion of both ingested food and digested food of third instars was found to be
significantly different among tomato genotypes with differing PPO activity levels, suggesting that PPO activity
rendered foliage less nutritious. Other studies in tomato have shown the role of PPO in disease resistance; antisense
suppression of PPO increases susceptibility, and PPO overexpression increases resistance, to Pseudomonas syringae
pv. tomato (Li and Steffens, 2002; Thipyapong et al., 2004a).
TABLE 10.1 Polyphenol Oxidase Activity by Wounded Cashew Apple
Specific Activity
Enzyme Extract (U/min/mg Protein) Activation
0 hour (control) 0.62 e
2�C/24 hours 2.92 4.8
27�C/24 hours 3.33 5.4
40�C/24 hours 1.07 1.7
Reprinted from Queiroz et al. (2011). � 2011, with permission from Elsevier.
mailto:Image of FIGURE 10.4|eps
FIGURE 10.5 (AeC) Second instar, (DeF) third instar and (GeI) fourth instar larvae of Spodoptera litura (F.) feeding on node 8 leaves of
tomato plants with varied polyphenol oxidase (PPO) activity levels. A14-6: transgenic line with suppressed PPO activity; NT: non-transformed
control; S-28: transgenic line overexpressing PPO activity. Scale bars¼ 2.5 mm (Reprinted from Mahanil et al., 2008). � 2008, with permission from
Elsevier.
393II. Characteristics of Polyphenol Oxidase
D. Phenolic Compounds in Food Material
Phenolic compounds are natural substances that contribute to the sensorial properties (color, taste, aroma, and
texture) associated with fruit quality (Marshall et al., 2000). They form one of the main classes of secondary
metabolites, with a large range of structures and functions, but generally possessing an aromatic ring bearing one or
more hydroxy substituents. The phenolic composition of fruits is determined by genetic and environmental factors
but may be modified by oxidative reactions. They are synthesized during plant development, but their synthesis is
stimulated in stress conditions by activation of phenylalanine ammonia-lyase. Thus, these compounds play a role in
defense and adaptive mechanisms of plants. The phenolic compounds which occur in food material and which
participate in browning may be classified into four groups, namely, simple phenols, phenolic acids, cinnamic acid
derivatives, and flavonoids.
394 Chapter | 10 Enzymatic Browning
1. Simple Phenols
The simple phenols include monophenols such as L-tyrosine and o-diphenols such as catechol, resorcinol, and
hydroquinone. However, among these compounds only catechol can be oxidized by PPO, because hydroxyl is at the
ortho position. Catechol was identified in Diospyro kaki roots by Jeong et al. (2009) and PPO showed high affinity
towards this phenolic compound (Özen et al., 2004).
OH
OH
OH
OH
catechol resorcinol hydroquinone
OH
OH
2. Phenolic Acids
This class comprises the acids synthesized from precursor benzoic acid and they are widely distributed in plants.
Gallic acid is present in an esterified form in tea flavonoids. Gallic and protocatechuic acids were found in cashew
apple (Michodjehoun-Mestres et al., 2009; Queiroz et al., 2011) and protocatechuic was the main free phenolic acid
identified in medlar fruits (Gruz et al., 2011).
Gallic acid R1=R2=R3=OH
Protocatechuic acid R1=H, R2=R3=OH
Vanillic acid R1=H, R2=OH, R3=OCH3
Syringic acid R2=OH, R1=R3=OCH3
Benzoic Acids
COOH
R1
R2
R3
3. Cinnamic Acid Derivatives
The most important member of this group of compounds in food material is chlorogenic acid, which is the key
substrate for enzymatic browning, particularly in apples and pears (Gauillard and Forget, 1997; Song et al., 2007).
Although potato is rich in chlorogenic acid, it is not a determinant factor for blackspot development. Two potato
cultivars (cv. ‘Bildtstar’ and cv. ‘Lady Rosetta’) were bruised and the pigments identified. Quinic acid was detectable
in hydrolysates of the pigments from ‘Bildtstar’, but not in those of ‘Lady Rosetta’, which indicated that chlorogenic
acid may take part in blackspot formation, but is not essential for the discoloration (Stevens and Davelaar, 1996).
These data were confirmed by Lærke et al. (2002), who found a correlation between black end products and free
tyrosine, but none between black color and chlorogenic and caffeic acids in potato cultivars (cv. ‘Dali’ and cv.
‘Oleva’). Discoloration derived from chlorogenic acid is attributed to the oxidation of complexes formed between
iron and caffeic and chlorogenic acids.
mailto:Image of FIGURE 10.5|tif
395II. Characteristics of Polyphenol Oxidase
Other members of thisgroup of compounds include p-coumaric, caffeic, ferulic, and sinapic acids. The universal
distribution and high concentration of cinnamic acids in fruits may be due to their function as precursors in the
biosynthesis pathway of more complex polyphenols.
4. Flavonoids
This group is the most widespread and structurally diverse among polyphenols. All members of this group of
compounds are structurally related to flavone:
In food material, the important flavonoids are the catechins, anthocyanins, and flavonols. Catechin has the
following structure:
mailto:Image of FIGURE 10.5|tif
396 Chapter | 10 Enzymatic Browning
An extra hydroxyl group attached to the 50 position on the B ring of catechin and epicatechin gives rise to gal-
locatechin and epigallocatechin, respectively. The catechin gallates are esters of catechins and gallic acid, the ester
linkage being formed from the carboxyl group of gallic acid and the hydroxyl group attached to position 3 of the
catechin C ring. An example is (�)-epigallocatechin gallate, which is the major polyphenol in dried tea leaves.
HO
CH
OH
OH
OH
C
HOH
O
O CO
OH
OH
OH
Such compounds are not the major phenols in fruits; however, they are important constituents of fruits in olig-
omeric or polymeric forms as proanthocyanidins or condensed tannins. Procyanidin is a dimer and is present in apple,
grape, and cherry (Robards et al., 1999).
HO
OH
OH
Procyanidin B-1
O
HO
OH
O
OH
OH
OH
OH
OH
The flavonols also participate in browning reactions and are widely distributed in plant tissues. The most
commonly occurring flavonols are kaempferol, quercetin, and myricetin:
HO
OH Kaempferol (R1 = R2 = H)
Quercetin (R1 = OH; R2 = H)
Myricetin (R1 = R2 = OH)
OH
OH
O
O
R1
R2
Flavonols possess a light yellow color and are particularly important in fruits and vegetables in terms of the
astringency they impart to the particular food. They occur naturally as glycosides, examples of which are rutin and
the glycosides of quercetin, the latter occurring in tea leaves and apple skins (Hulme, 1958).
All of the compounds discussed so far are substrates for PPO. Such oxidation reactions are important in tea
fermentation, in the browning of cling peaches (Luh et al., 1967), and in the drying stage of the curing of fresh cacao
seeds (Roelofsen, 1958). It appears to be an important step in the development of the final color, flavor, and aroma of
cacao and chocolate. PPO plays a beneficial role in the case of tea and cacao fermentation, in contrast to its role in the
browning of fruits and vegetables.
mailto:Image of FIGURE 10.5|tif
mailto:Image of FIGURE 10.5|tif
1.2
1
0.8
0.6
0.4
0 0.5 1
Ab
so
rb
an
ce
 a
t 5
10
 n
m
1.5
Time (h)
3.52 2.5 3
FIGURE 10.6 Changes in absorbance of bayberry anthocyanin at 510 nm during its degradation by bayberry polyphenol oxidase (PPO) in the
presence (:) and absence (D) of gallic acid and after thermal denaturation of the PPO in the presence of gallic acid (-) (Reprinted from Fang
et al., 2007).� 2007, with permission from Elsevier.
397II. Characteristics of Polyphenol Oxidase
Several studies have been conducted to identify other endogenous substrates for PPO in fruits. Epicatechin is the
endogenous substrate for litchi (lychee) and longan PPOs (Sun et al., 2006; Shi et al., 2008). Other flavonoids, such as
eriodictyol, myricetin, and fisetin, can also be oxidized by PPO (Jiménez et al., 1998; Jiménez and Garcı́a-Carmona,
1999; Jiménez-Atiénzar et al., 2005a, b). Anthocyanins are not directly oxidized by PPO or are poor substrates
(Mathew and Parpia, 1971), but they could be degradated by a coupled oxidation mechanism. Ruenroengklin et al.
(2009) suggested that litchi PPO directly oxidized epicatechin, then oxidative products of epicatechin, in turn,
catalyzed litchi anthocyanin degradation, and finally resulted in the browning reaction, which can account for
pericarp browning of postharvest litchi fruit. Similar results were found by Fang et al. (2007) which demonstrated
that the rate of anthocyanin degradation in bayberry was stimulated by the addition of gallic acid. In the absence of
gallic acid, the rate of cyaniding 3-glucoside discoloration was almost the same as when the enzymatic extract was
inactivated by heating and gallic acid was present (Figure 10.6).
E. Laccase
Early studies on enzymatic browning of phenolic compounds identified two types of activity which were initially
termed tyrosinase and laccase. The respective systematic names formerly used were o-diphenol: oxygen oxidore-
ductase (EC 1.10.3.1) and p-diphenol: oxygen oxidoreductase (EC 1.10.3.2), which are now combined as mono-
phenol monooxygenase (EC 1.14.18.1). The diagnostic feature of laccase is its ability to oxidize p-diphenols,
a property not possessed by tyrosinase or PPO (Mayer and Harel, 1979). It is present in many plants, fungi, and
microorganisms; however, most of the known laccases are of fungal origin, in particular from the white rot fungi
mailto:Image of FIGURE 10.6|eps
mailto:Image of FIGURE 10.6|eps
398 Chapter | 10 Enzymatic Browning
(Thurston, 1994; Revankar and Lele, 2006; Fonseca et al., 2010). The basic laccase-catalyzed reaction responsible
for the oxidation of p-diphenol is:
Laccase is responsible for the oxidation of flavonoids and browning during Arabidopsis seed development
(Figure 10.7) and is one of the major enzymes responsible for oxidation of lignin, an amorphous polymer which
functions as a cementing material in wood cells. The enzyme acts in cooperation with other ligninolytic (lignin-
degrading) fungal enzymes such as lignin peroxidase, manganese peroxidase, and versatile peroxidase (Arora et al.,
2002). Laccase alone can only oxidize the phenolic lignin units. Therefore, laccase is often applied with an oxidation
mediator, a small molecule able to extend the effect of laccase to non-phenolic lignin units and to overcome the
accessibility problem (Srebotnik and Hammel, 2000; Shleev et al., 2006). The mediator is first oxidized by laccase and
then diffuses into the cell wall, oxidizing lignin inaccessible to laccase. The use of these mediators in laccase reactions
enables the application of the enzyme in the forest industry, removing pitch, phenolic contaminants, and dyes from
wood-based materials and water; laccase technology is applicable to virtually the entire production chain of paper
products from pulping to recovery of secondary fibers and effluent treatment (Widsten and Kandelbauer, 2008).
F. Specificity of Polyphenol Oxidase
As discussed previously, PPO catalyzes two different reactions, either the hydroxylation of monophenols to
o-dihydroxyphenols or the oxidation of o-dihydoxyphenols to o-quinones. The most appropriate chemical
structure with respect to PPO activity, when the reaction rate is at a maximum, appears to correspond to the
o-dihydroxy structure as evident in such compounds as catechol, caffeic acid, and the catechins (Rocha and
Morais, 2001; Gawlik-Dziki et al., 2007). Oxidation of o-diphenols to the corresponding o-quinones is a general
reaction of all known PPO, irrespective of whether the source material is potato, sweet potato (Hyodo and Uritani,
1965), lettuce (Gawlik-Dziki et al., 2008), apple (Harel et al., 1966), tomato (Hobson, 1967), banana (Ünal,
2007), artichoke (Aydemir, 2004), tobacco (Shi et al., 2002), cashew apple (Queiroz et al., 2011), litchi
(Ruenroengklin et al., 2009; Yue-Ming et al., 1997), or green olives (Segovia-Bravo et al., 2009). Monophenols
FIGURE 10.7 Seed coat pigmentation in Arabidopsis: illustration of a browning process. (a) Photographs showing the appearance of a brown
pigmentation in the testa of the wild-type genotype during seed desiccation. The brown pigment is absent from the transparent testa 10 (tt10) mutant defective
in a laccase enzyme. (b) Mutant tt10 seeds slowly become brown after harvest and eventually resemble wild-type seeds. Scale bar¼ 550mm. (c) Schematic
drawing indicating the occurrence of brown pigmentation duringArabidopsis seed development. DAF: days after flowering (Reprint from Pourcel et al.,
2006).� 2006, with permission from Elsevier.
399II. Characteristics of Polyphenol Oxidase
are more slowly acting substrates as they have to be hydroxylated before their oxidation to the corresponding
o-quinones. The oxidation of monophenols is less widespread than that of diphenols, being catalyzed, for
example, by potato and mushroom enzyme preparations. The relationship between cresolase and catecholase
activities is not yet fully understood. It appears that many PPOs are specific to a high degree in that they attack
only o-diphenols. Aydemir (2004), in a study on artichoke PPO, found that it exhibited the highest specificity
towards catechol, followed by 4-methycatechol and pyrogallol. According to Espı́n et al. (1998), the best
substrates are those with low molecular size substituent side-chain and high electron donor capacity. Erat et al.
(2006), studying PPO activity in Ferula sp., found the best results using catechol and (�)-epicatechin with
enzyme extracted from leaf and stem, respectively. Sellés-Marchart et al. (2006) found chlorogenic acid to be the
most efficient substrate, followed by 4-methylcatechol, 4-tert-butylcatechol, epicatechin, catechol, and isopro-
terenol, for loquat fruit (Eriobotrya japonica Lindl.). PPO from Henry chestnuts catalyzed the oxidation of
catechol and pyrogallic acid, but had no effect on cresol or tyrosine (Xu et al., 2004). The substrate with the
highest activity, for Amasya apple (Mallus sylvestris Miller cv. Amasya) PPO, was catechol, followed by
4-methylcatechol, pyrogallol, and 3,4-dihydroxylphenylalanine (L-DOPA) (Oktay et al., 1995). Other kinetic
parameters found by researchers are described in Table 10.2.
TABLE 10.2 Characteristics of Polyphenol Oxidases from Plant Sources
Optimum
Plant Substrate Km (mM) temperature (�C) Optimum pH
Applea Catechol 34 15 7.0
4-Methylcatechol 3.1
Artichokeb Catechol 10.2 25 6.0
4-Methylcatechol 12.4
Bananac Catechol 8.5 30 7.0
Broccolid Catechol 12.3 e 5.7
4-Methylcatechol 21.0
Cashew applee Catechol 18.8 e 6.5
Grapef Chlorogenic acid 3.2 25 5.0
Catechin 4.3
Mangog Catechol 6.3 30 7.0
Melonh DOPAC l 7.2 60 7.0
Strawberryi Catechol 5.9 25 5.0
Vanillaj Catechol 85.0 37 3.4
4-Methylcatechol 10.6 37 3.0
Yacon rootk Caffeic acid 0.2 30 6.6
Chlorogenic acid 1.1
aOktay et al. (1995)
bAydemir et al. (2004)
cÜnal (2007)
dGawlik-Dziki et al. (2007)
eQueiroz et al. (2011)
fRapeanu et al. (2006)
gWang et al. (2007)
hChisari et al. (2008)
iDalmadi et al. (2006)
jWaliszewski et al. (2009)
kNeves and Silva (2007)
l3,4-dihydroxyphenylacetic acid.
Adapted from Queiroz et al. (2008).
400 Chapter | 10 Enzymatic Browning
III. POLYPHENOL OXIDASE IN FOODS AND FOOD PROCESSING
A. Role in Tea Fermentation
The production of black tea is dependent on the oxidative changes that Camellia sinensis leaf polyphenols undergo
during processing. Such changes are particularly important for the development of color as well as the reduction of
the bitter taste associated with unoxidized tannin (polyphenol compound). Several types of tea, namely white, green,
oolong, and black tea, come from the same plant, C. sinensis. It is the processing that the tea leaves go through which
determines what type they will become and will also alter the phenolic composition of the tea.
The main tea leaf polyphenols, determined by partition chromatography, include (þ)-catechin, (�)-epicatechin,
(þ)-gallocatechin, (�)-epigallocatechin, (�)-epicatechin gallate, and (�)-epigallocatechin gallate. Of these
compounds, (�)-epigallocatechin gallate is the major component in the tea shoot. During tea processing, Muthumani
and Kumar (2007) observed that considerable quantities of epigallocatequin gallate, epigallocatechin, and epi-
catechin gallate were oxidized to form theaflavins and their gallates. Changes in catechin and epicatechin contents
were not observed, probably owing to the formation of free gallic acid and catechin from the other catechin fractions
such as epigallocatequin gallate, epigallocatechin, and epicatechin gallate, by oxidative degallation. Different results
were published by Munoz-Munoz et al. (2008). These authors found that the best substrates for PPO in tea leaves are
epicatechin followed by catechin, owing to better access of enzyme to hydroxyl radicals.
The production of black tea, the most popular form of the beverage, is carried out in four stages. The first stage is
called withering, when the shoots from the tea plant are allowed to dry out. This is followed by rolling with a roller,
which disrupts the tea leaf tissue and causes cell damage, providing the necessary conditions for the development of
the oxidative processes. The next step is the fermentation of the fragmented tea leaves, which are held at room
temperature in a humid atmosphere with a continuous supply of oxygen. These conditions are optimal for PPO action
on the tea leaf catechins, which, in addition to reducing astringency, converts the green color of the rolled tea leaves to
give coppery-red and brown pigments. Fermentation is terminated by firing, where the tea is dried at 90e95�C and
the moisture reduced to 3e4%.
The critical biochemical reaction during tea fermentation is oxidation of catechins by PPO to the corresponding
o-quinones. These quinones are intermediate compounds which are subjected to secondary oxidation leading to the
production of theaflavin and theaflavin gallate, the yelloweorange pigments in black tea, and to a group of
compounds referred to as thearubigins. These thearubigins are dark brown and the main contributors to the familiar
color of black tea, and they are the oxidative products of theaflavins. A simplified scheme for the oxidative reactions
occurring during tea fermentation is outlined in Scheme 10.2. The theaflavin content of tea was shown by Hilton and
Ellis (1972) to correlate with the tea taster’s evaluation. This was consistent with earlier studies by Roberts (1952)
and Sanderson (1964), who noted a positive correlation between tea quality and PPO activity. The enzyme was later
purified by Hilton (1972). The oxidative degradation of phloroglucinol rings of the theaflavins by peroxidase caused
a loss of theaflavins and a decline in tea quality (Cloughley, 1980a, b). Consequently, the presence of both these
enzymes affects the quality of tea. Van Lelyveld and de Rooster (1986) examined the browning potential of black tea
clones and seedlings. They found a much higher level of PPO in a high-quality hybrid clone (MT12) compared to
a low-quality seedling tea (Table 10.3). The reverse was true for peroxidase, in which lower quality tea had more than
double the activity of peroxidase. This suggested that the combination of higher theaflavin levels and PPO activity
was responsible for the better quality associated with the MT12 clone. Subramanian et al. (1999) studied the role of
PPO and peroxidase in the formation of theaflavins. They demonstrated that PPO generates hydrogen peroxide
(H2O2) during oxidation of catechins and peroxidase utilizes the formed H2O2 for subsequent oxidation of the
products of PPO-catalyzed reactions, decreasing the content of theaflavins. The fermentation time is also important to
the formation of theaflavins and thearubigins. Muthumani and Kumar (2007) reported an optimum time of 45
minutes, when theaflavins reached their maximum content; after this time their content declined to 20%, leading to
a lower quality tea.
SCHEME 10.2 Oxidative transformations of (�)-epigallocatechin and its gallate during tea fermentation.
TABLE 10.3 Specific Activity (D OD/min/mg protein) of MT12 and Seedling Tea Leaves
Clone Peroxidasea Polyphenol Oxidaseb
M12 1.821 0.055
Seedling 0.735 0.020
aSignificant at p < 0.05
bSignificant at p < 0.01.
From Van Lelyveld and de Rooster (1986).
401III. Polyphenol Oxidase in Foods and Food Processing
Green tea is particularly popular in Oriental countries, such as Japan. It is an unfermentedtea with a light color
and a characteristic degree of astringency owing to a high content of catechins. This is achieved by the application of
heat during the early stages of tea manufacture, which inhibits or prevents oxidation. Red and yellow teas are
intermediate between black and green teas and are semifermented products (partially fermented before firing). An
example of the latter is the Chinese variety oolong. Tea leaf catechins generate theaflavins and thearubigins during
the longer processing time. White and green teas have higher levels of catechin, whereas oolong and black teas are
rich in theaflavins and thearubigins (Table 10.4).
B. Shrimps and Crustaceans
Enzymatic browning, having been studied extensively in fruits and vegetables, has also been implicated in the
discoloration, called melanosis or blackspot, of shrimps and other crustaceans (Zamorano et al., 2009; Giménez
et al., 2010), which renders these products unattractive to the consumer and lowers their market value. In crustaceans,
PPO is mainly located in the cuticle, specifically on the internal surface, inside chromatophores. During iced storage
of crustaceans, the inactive PPO stored in homocytes and digestive glands can also be activated by the action of
proteolytic enzymes leaching from the digestive tract. Moreover, protein hydrolysis by these proteases provides
substrates for active PPO (Ali et al., 1994).
A recent study by Zamorano et al. (2009) evaluated PPO activity in situ and in partially purified extracts from
different organs of deepwater pink shrimp (Parapenaeus longirostris). They found higher enzyme activity in cara-
pace extracts, but marked melanosis developed on the cephalothorax and head after 1 day at 4�C (Figure 10.8). Even
after 7 days at 4�C, there was no melanosis in the carapace, confirming that the development of melanosis in the
different tissues depends on another factor in addition to PPO levels. In native non-denaturating conditions, a band of
500 kDa was identified which was capable of oxidizing dihydroxyphenylalanine. Table 10.5 shows some biochemical
characteristics of PPO extracted from marine sources.
TABLE 10.4 Flavonoid Composition of Tea (Percent by Dry Weight)
Component Green Tea Black Tea
Total flavonoids 15e25 15e25
Total catechins 12e18 2e3
(�)-Epicatechin 1e3 < 1
(�)-Epicatechin gallate 3e6 < 1
(�)-Epigallocatechin 3e6 < 1
(�)-Epigallocatechin gallate 9e13 1e2
Flavonols 2e3 1e2
Theaflavins < 1 4
Other polyphenols 2e4 7e15
Reprinted from Hodgson and Croft (2010). � 2010, with permission from Elsevier.
FIGURE 10.8 Melanosis appear-
ance in different anatomical parts of
deepwater pink shrimp during 0, 1,
4, and 7 days of storage at 4�C. (A)
Whole shrimp; (B) Whole shrimp with
the carapace removed; (C) Head
(carapace þ cephalothorax þ pereo-
pods and maxillipeds); (D) Carapace;
(E) Head with the carapace removed;
(F) Pereopods þ maxillipeds; (G)
Whole abdomen (including the
exoskeleton, muscle, pleopods, and
telson); (H) Abdomen with the muscle
removed (left) and corresponding
muscle (right); and (I) Telson
(Reprinted from Zamorano et al.,
2009). � 2009, with permission from
Elsevier.
402 Chapter | 10 Enzymatic Browning
Melanosis inhibitors are used in marine food to prevent browning and extend the shelf-life of products. Traditional
methods include the use of sulfite-based formulations, but sulfite causes adverse reactions. Therefore, other
compounds, including those of natural origin, have been studied as alternatives to sulfur compounds. Martı́nez-
Alvarez et al. (2007) sprayed 4-hexylresorcinol-based formulations on Norway lobsters. After 12 days of storage they
TABLE 10.5 Characteristics of Polyphenol Oxidases from Marine Sources
Optimum
Crustacean Substrate Km temperature (�C) Optimum pH
Deepwater pink shrimpa DOPA 1.85 15e60 4.5
Prawnb DOPA e 40e60 5.0 and 8.0
Norway lobsterc
(carapace)
Catechol 19.40 60 7.0
Norway lobsterc
(viscera)
Catechol 5.90 60 8.0
DOPA: 3,4-dihydroxylphenylalanine.
aZamorano et al. (2009)
bMontero et al. (2001)
cGiménez et al. (2010).
403IV. Control or Inhibition of Enzymatic Browning
observed lower PPO activity and melanosis and improvement in the appearance of the lobsters. Cystein and
glutathione inhibited the oxidation of L-DOPA catalyzed by kuruma prawn PPO (Benjakul et al., 2006). Among
natural substances, recent studies have demonstrated the potential use of phenolic compounds inhibiting browning.
Shrimps treated with 2% ferulic acid had, after 10 days of storage, a lower melanosis score and higher scores for
color, flavor, and overall likability, compared with the control and sodium metabisulfite-treated shrimps (Nirmal and
Benjakul, 2009). Treatment combining catechin and ferulic acid before freezeethawing was efficient in retarding
melanosis in Pacific white shrimp. Phenolics also acted to decrease the growth of psychrophilic bacteria and retard
lipid oxidation during the subsequent refrigerated storage (Nirmal and Benjakul, 2010).
Novel technology such as high hydrostatic pressure was studied by Montero et al. (2001), achieving partial
inactivation of prawn PPO after treatment at 300e400MPa for 10 minutes. The mechanism of enzyme inactivation
by high pressure is discussed in Section IV, D.
IV. CONTROL OR INHIBITION OF ENZYMATIC BROWNING
The inactivation of PPO is required to minimize product losses caused by browning. Several methods and tech-
nologies have been studied. Heat treatment and addition of antibrowning agents are usually applied, but several
researchers have proposed the application of other methods as alternatives to thermal processing for PPO inactivation
(Queiroz et al., 2008).
Inhibitors of PPO activity can be based on their mode of action, for example, the exclusion of reactants such as
oxygen, denaturation of enzyme protein, interaction with the copper prosthetic group, and interaction with phenolic
substrates or quinones.
A. Exclusion of Oxygen
The simplest method of controlling enzymatic browning is by immersing the peeled product, such as potato, in water
before cooking. This can be done very easily in the home to limit access of oxygen to the cut potato tissue. This
procedure has been used for a long time on a large scale for the production of potato chips and French fries (Talburt
and Smith, 1967). The method is limited as the fruit or vegetable will brown on re-exposure to air or via the oxygen
occurring naturally in the plant tissues. The removal of oxygen from fruit or vegetable tissue could lead to
anaerobiosis if it is stored for extended periods, which in turn could lead to abnormal metabolites and tissue
breakdown. In the case of frozen sliced peaches, the surfaces are treated with excess ascorbic acid to use up the
surface oxygen.
More recently, modified atmospheres have also been used to prevent enzymatic browning. Since browning is an
oxidative reaction, it can be retarded by eliminating oxygen from the cut surface of the vegetables (Limbo and Pier-
giovanni, 2006). Modified atmosphere packaging has the potential to extend the shelf-life of fruits and vegetables,
mainly by limiting the oxidation processes. Low-oxygen and elevated carbon dioxide atmospheres can reduce surface
404 Chapter | 10 Enzymatic Browning
browning. This decrease in the browning phenomenon is accompanied by several physiological effects such as
a decrease in respiration rate and a delay in the climacteric onset of the rise in ethylene (Solomos, 1997). Day (1996)
hypothesized that high oxygen concentrations may cause substrate inhibition of PPO or, alternatively, high levels of
colorless quinones formed may cause feedback inhibition of the enzyme. Gorny et al. (2002) found that low-oxygen
(0.25 or 0.5 kPa), elevated carbon dioxide (air enriched with 5, 10, or 20 kPa CO2), or high-oxygen (40, 60, or 80 kPa)
active atmospheres alone did not effectively prevent surface browning of fresh-cut pear slices. Studies on the influence
of modified atmosphere packaging and the use of antioxidantcompounds on the shelf-life of fresh-cut pears have
mainly focused on the sensory qualities of the commodity (Sapers and Miller, 1998; Gorny et al., 2002; Soliva-Fortuny
et al., 2002, 2004). According to Oms-Oliu et al. (2008), knowledge about the impact of dipping treatments and
packaging conditions on antioxidant properties of fresh-cut pears is still incomplete, especially in regard to high-oxygen
active packaging. Y. Kim et al. (2007) evaluated the effects of hot water treatment in combination with controlled
atmosphere (CA) storage and the resultant changes in quality and antioxidant polyphenolics in mango. They observed
that the external quality of mango fruit during ripening was greatly improved by low-oxygen and/or high-carbon
dioxide storage conditions and hot water immersion treatments. Carbon dioxide storage delayed fruit ripening, as
evidenced by physicochemical changes, and the hot water treatment plus CA storage was an effective treatment
combination to extend the postharvest life of mangoes, without adversely changing the nutritional profile of the fruit.
Limbo and Piergiovanni (2006) studied the effects of high oxygen partial pressures in combination with ascorbic
and citric acid on the development of the enzymatic browning of peeled and cut potatoes (‘Primura’ variety),
packaged in flexible pouches and stored at 5�C for 10 days. They observed that the treatments with high oxygen
partial pressures had some positive effects on enzymatic browning only if the initial atmosphere was close to 100 kPa
and the dipping acid concentrations were chosen with care. As Wszelaki and Mitcham (2000) have shown, near
100 kPa oxygen atmospheres could be difficult to maintain either in a package or on a larger scale, as well as being
dangerous owing to flammability. A study conducted by Saxena et al. (2008) showed that the synergistic effect of
antibrowning and antimicrobial compounds with reduced oxygen and elevated carbon dioxide atmosphere could
enhance the keeping quality of fresh-cut jackfruit bulbs by minimizing deteriorative changes in physiological,
sensorial, and microbial attributes. Martı́nez-Sánchez et al. (2011), studying the influence of oxygen partial pressures
(pO2) and light exposure during storage on the shelf-life of fresh-cut Romaine lettuce, noticed that the consumption
of oxygen in samples exposed to 24-hour light differed significantly from that of those stored in a 12-hour light/12-
hour dark photoperiod or in 24-hour darkness (10.6� 7.0, 18.3� 3.5, and 25.8� 8.6 nmol O2/kg/second, respec-
tively). Packages exposed to light showed higher pO2 than packages stored in darkness, while those exposed to the
photoperiod had intermediate values. This study showed that under light conditions respiration activity was
compensated by photosynthesis, resulting in a higher pO2. Thus, browning of fresh-cut Romaine lettuce can be
promoted by light exposure during storage as it increases headspace pO2. In fact, uncontrolled light conditions during
storage and commercial distribution can contribute to the induction of the browning process, which can cause the
product to be unsaleable.
B. Chemical Inhibitors of Polyphenol Oxidase
Different effectors can control enzymatic browning and these compounds are classified, based on the inhibition
mechanism, as reducing agents, chelating agents, acidulants, enzyme inhibitors, enzyme treatments, and complexing
agents (Özo�glu and Bayındırlı, 2002).
Several compounds possess chemical structures closely related to o-diphenols but do not function as substrates of
PPO. These include methyl-substituted derivatives such as guaiacol and ferulic acid (Finkle and Nelson, 1963;
Nirmal and Benjakul, 2009), and m-diphenols such as resorcinol and phloroglucinol, which have been shown to have
an inhibitory effect on PPO activity, either in a competitive way (Montero et al., 2001; Waliszewski et al., 2009) or by
a slow-binding inhibition mechanism (Jiménez and Garcı́a-Carmona, 1997), depending on the substrate.
405IV. Control or Inhibition of Enzymatic Browning
Since PPO is a metalloprotein in which copper is the prosthetic group, it is inhibited by a variety of chelating
agents, including sodium diethyldithiocarbamate (DIECA), sodium azide, potassium ethylxanthate, and ethylene-
diaminetetraacetate (EDTA). While sodium azide and EDTA inhibit PPO, they appear to be less specific chelators
than DIECA or potassium ethylxanthate (Kavrayan and Aydemir, 2001; Aydemir, 2004; Gawlik-Dziki et al., 2007;
Pinto et al., 2008; Gao et al., 2009). The latter compounds were reported by Anderson (1968) to combine with the
quinones produced by PPO in addition to chelating copper.
Sulfites are strong inhibitors and have been used for a long time in the food industry. However, an excess of sulfite-
based formulations can cause adverse reactions, mainly in asthmatic people. Since it was reported by the 51st
meeting of the Joint Expert Committee on Food Additives that the intake of sulfites could be above safe limits, their
use as food additives is not recommended. Researchers have been carrying out studies to find potent antibrowning
agents which cause no damage to the organism. L-Cysteine has been shown to be an effective inhibitor of PPO by
acting as a quinone coupler as well as a reducing agent. It reacts with o-quinone intermediates to produce stable and
colorless products (_Iyido�gan and Bayındırlı, 2004). Since Kahn (1985) reported L-cysteine to be the most effective
inhibitor of avocado, banana, and mushroom PPO, many studies have shown its effect on the PPO activity of arti-
choke, apple juice, mango purée, grape, and lettuce (_Iyido�gan and Bayındırlı, 2004; Guerrero-Beltrán et al., 2005;
Rapeanu et al., 2006; Altunkaya and Gökmen, 2009). However, effective concentrations of cysteine produce an
undesirable odor, limiting its use in food processing. Altunkaya and Gökmen (2009), trying to select the most
effective antibrowning compound on fresh lettuce, evaluated the effectiveness of ascorbic acid, cysteine, citric acid,
and oxalic acid. They found that lettuce treated with oxalic acid and ascorbic acid maintained a higher level of
phenolic compounds than citric acid and cysteine. Interestingly, cysteine had no positive effect on the prevention of
oxidation of phenolic compounds even though it prevented browning in lettuce.
The application of acids to control enzymatic browning is used extensively. The acids employed are those found
naturally in plant tissues, including citric, malic, phosphoric, and ascorbic acids. This method is based on the fact that
lowering the tissue pH will reduce or retard the development of enzymatic browning. The optimum pH of most PPO
lies between pH 4.0 and 7.0, with little activity below pH 3.0, as illustrated in Table 10.2. Muneta (1977) examined
the effect of pH on the development of melanins during enzymatic browning. Although, as discussed earlier, the
initial reaction involving the formation of quinone is enzyme catalyzed, polymerization of these quinones to the
brown or browneblack melanins is essentially non-enzymatic. Since both are pH dependent, Muneta (1977) studied
the effect of pH on the non-enzymatic reactions leading to the formation of melanins in potatoes. The formation of
dopachrome from dopaquinone was monitored in phosphate buffers at pH 5.0, 6.0, and 7.0. Very rapid melanin
development occurred at pH 7.0, compared to a rather slow process at pH 5.0. This observation could be important to
the processor, particularly if the lye-peeled potatoes are inadequately washed, resulting in a high surface pH that
facilitates enzymatic browning and melanin formation.
Citric acid has been used in conjunction with ascorbic acid or sodium sulfite as a chemical inhibitor of enzymatic
browning (Limbo and Piergiovanni, 2006; Ducamp-Collin et al., 2008; Hiranvarachat et al., 2011; Queiroz et al.,
2011). Cut fruit or vegetables are often immersed in dilute solutions of these acids just before processing. This is
particularly importantin the case of lye-peeled cling fruits, where the acid dip counteracts the effect that any residual
lye might have on enzymatic browning. Citric acid also inhibits PPO by chelating the copper moiety of the enzyme.
Compared to citric acid, however, malic acid, the principal acid in apple juice, is a much more effective inhibitor of
enzymatic browning.
A particularly effective inhibitor of PPO is ascorbic acid (Özo�glu and Bayındırlı, 2002). It does not have
a detectable flavor at the level used to inhibit this enzyme, nor does it have a corrosive action on metals. This
vitamin acts as an antioxidant because it reduces the quinone produced before it undergoes secondary reactions
that lead to browning, and also decreases the pH for the enzyme activity (Guerrero-Beltrán et al., 2005). The
mode of action of ascorbic acid is outlined in Scheme 10.3 (Walker, 1976). Ascorbic acid can inhibit the
enzyme by decreasing pH and acting as an antioxidant. Thus, it reduces quinones to diphenols, reversing
the reaction and decreasing brown pigments. Degl’Innocenti et al. (2007) evaluated lettuce, escarole, and rocket
salad and their sensitivity to enzymatic browning. They observed that the resistance of rocket salad to browning
was associated with the highest ascorbic acid content compared to other species. A study by Hsu et al. (1988)
compared the effectiveness of several ascorbic acid derivatives, including dehydroascorbic acid, isoascorbic
acid, ascorbic acid-2-phosphate, and ascorbic acid-2-sulfate, with that of ascorbic acid. On the basis of kinetic
studies they found ascorbic acid and isoascorbic acid to be the most effective inhibitors of mushroom PPO,
followed by dehydroascorbic acid. According to Özo�glu and Bayındırlı (2002), ascorbic acid has been shown to
SCHEME 10.3 Reduction by ascorbic
acid of the primary quinone oxidation
products of enzymatic browning (Walker,
1976).
406 Chapter | 10 Enzymatic Browning
be more effective than its isomer isoascorbic acid. The effect of ascorbic acid can be considered temporary
because it is oxidized irreversibly by reaction with intermediates, such as pigments, endogenous enzymes, and
metals such as copper. In apple juice, an inhibitory effect of ascorbic acid at a concentration of 1.8 mM was
observed for 4 hours.
An adequate amount of ascorbic acid must be added to food material to delay enzymatic browning. In fruit juices
treated with ascorbic acid, autooxidation of ascorbic acid, or natural ascorbic acid oxidase activity, will use up any
dissolved oxygen in the fruit juice. Thus, oxygen would become the limiting factor determining the rate of enzymatic
browning. The addition of ascorbic acid at a concentration of 300 mg per pound (0.454 kg) of fruit was shown by
Hope (1961) to control browning as well as reduce headspace oxygen in canned apple halves. This method was
particularly effective in controlling enzymatic browning in spite of the thickness of the apple tissue and its
comparatively high oxygen content.
Muneta (1977) suggested sodium acid pyrophosphate as an alternative inhibitor of enzymatic browning. It has
several advantages over the organic acids, being much less sour than citric acid, as well as minimizing after-cooking
blackening of potatoes by complexing iron. An additional benefit of chelating iron is to limit its role in the catalysis of
rancidity in fried or dehydrated potatoes.
Kojic acid is found in many fermented Japanese foods. Although it is present in certain foods as a natural
fermentation product, the use of kojic acid in the food industry may be restricted by the difficulties of large-scale
production and its high cost. _Iyido�gan and Bayındırlı (2004) showed a significant antibrowning effect of kojic acid in
concentrations ranging from 1 to 4 mM. This agent inhibited PPO and bleached melanin through chemical reduction
of the browning pigment to colorless compounds.
Benzoic and cinnamic acids, aromatic carboxylic acids, are PPO inhibitors owing to their structural similarities
with the phenolic substrates. Undissociated forms of these acids are able to inhibit PPO through complexation with
copper at the active site of the enzyme (Marshall et al., 2000). Rapeanu et al. (2006) reported a weak inhibition at
0.5 mM (23%) when benzoic acid was tested in grapefruit.
407IV. Control or Inhibition of Enzymatic Browning
Cyclodextrins are a family of cyclic oligosaccharides composed of a-1,4-linked glucopyranose subunits.
Cyclodextrins are produced from starch by enzymatic degradation. These macrocyclic carbohydrates with apolar
internal cavities can form complexes with and solubilize many normally water-insoluble compounds (Singh et al.,
2002). b-Cyclodextrins bind substrate in its hydrophobic core. The most important functional property of cyclo-
dextrins is their ability to form inclusion complexes with a wide range of organic guest molecules, including PPO
substrates (Irwin et al., 1994). Because they can form inclusion complexes the properties of the materials with which
they complex can be modified significantly. As a result of molecular complexation phenomena cyclodextrins are
widely used in many industrial products, technologies, and analytical methods (Martin Del Valle, 2004). Özo�glu and
Bayındırlı (2002), using concentrations ranging from 0.3 to 1.8 mM, showed no effect of b-cyclodextrin, indicating
that a higher amount of the compound was necessary to achieve inhibition. In addition, PPO activity in apple juice
was affected by the type of cyclodextrin (López-Nicolás et al., 2007). To achieve efficiency in PPO inhibition, food
industries may pay attention to the major phenolic compounds present in the product and the type of b-cyclodextrins
used.
Sodium chloride is a strong oxidizing agent which can generate chlorine dioxide under acidic conditions. Below
pH values of about 5 a strongly pH-dependent inhibitory effect is observed and the degree of inhibition increases with
the acidity of the reaction medium. In contrast, an activation effect is observed at higher pH values (Valero and
Garcı́a-Carmona, 1998; M. H. Fan et al., 2005). Severini et al. (2003) evaluated the effect of sodium chloride on the
prevention of enzymatic browning in sliced potatoes and observed that all the considered blanching treatments
allowed PPO inactivation. With regard to color, the use of calcium chloride, at low concentrations, would seem better
than the use of sodium chloride.
These differences in the mechanisms allow the use of combinations of antibrowning agents that may result in
enhancement of inhibition. A combination of ascorbic acid, cysteine, and cinnamic acid showed a synergistic effect
compared to the individual compounds in cloudy apple juice (Özo�glu and Bayındırlı, 2002). _Iyido�gan and Bayındırlı
(2004) obtained 89% of enzymatic inhibition in Amasya apple juice treated with 3.96 mM L-cysteine, 2.78 mM, kojic
acid, and 2.34 mM 4-hexylresorcinol (4-HR). Also in apple juice, a synergic effect was observed between b-
cyclodextrin and 4-HR which was not observed for the combination of b-cyclodextrin and methyl jasmonate. In the
first combination, b-cyclodextrin reduced the concentration of free substrate that could be oxidized, while 4-HR
interacted directly with the enzyme by a competitive mechanism. In the second case, b-cyclodextrin complexed with
the substrate and methyl jasmonate, increasing the amount of free substrate and decreasing the concentration of
methyl jasmonate that could inhibit PPO activity (Alvarez-Parrilla et al., 2007). The inhibition by methyl jasmonate
observed in this study might be an exception, since some authors have described PPO activation by this compound
(Koussevitzky et al., 2004; Melo et al., 2006).
Some natural agents have an antibrowning effect. Honey contains a number of components that act as preser-
vatives, such as a-tocopherol, ascorbic acid, flavonoids, and other phenolic compounds. Honey from different floral
sources reduced PPO activity by 2e45% in fruit and vegetable homogenates.When combined with ascorbic acid, this
inhibitory effect is enhanced (Chen et al., 2000). It was also shown that native procyanidins, flavanol polymers that
occur naturally in plants, inhibited PPO activity in cider apple juices and the inhibition intensity increased with
degree of polymerization of the procyanidins. The mechanism is probably due to the binding of the polyphenol to the
protein, affecting the catalytic activity of the enzyme or by forming an inactive enzymeepolyphenolesubstrate
complex (Le Bourvellec et al., 2004). Lee and Park (2005) tested Maillard reaction products, synthesized from
various amino acids and sugar solutions, on potato PPO activity and observed an inhibitory effect which was
dependent on the amino acid (arginine> cysteine> histidine> lysine) and the type of sugar used (monosaccharides
> disaccharides).
Proteins, peptides, and amino acids can affect PPO activities by reacting with the o-quinones and by chelating
the copper at the active site of PPO. Girelli et al. (2004) measured PPO activity in the presence of various glycyl-
dipeptides and they found that these compounds can affect PPO activities by reacting with o-quinones and by
chelating the copper at the active site of PPO. Glycylaspartic acid, glycylphenilalanine, glycylglycine, glycylly-
sine, glycyltyrosine, and glycylhistidine affected the formation of quinone at all concentrations used, varying
from 1 to 50 mM. In a study conducted by Shi et al. (2005), different inhibition types between cinnamic acid
and its derivatives were demonstrated. Strength and inhibition types were: cinnamic acid (non-competitive) >
4-hydroxycinnamic acid (competitive) > 4-methoxycinnamic acid (non-competitive). No inhibitory effect of
2-hydroxycinnamic acid on the diphenolase activity was found. According to the authors, these inhibitors were
attached to a different region from the active site and hindered the binding of substrate to the enzyme through steric
408 Chapter | 10 Enzymatic Browning
hindrance or by changing the protein conformation. Chen et al. (2005) demonstrated that PPO was inhibited by
several p-alkoxybenzoic acids and that p-methoxybenzoic acid was the most potent inhibitor. Song et al. (2006)
studied the inhibitory effects of cis- and trans-isomers of 3,5-dihydroxystilbene. Although both compounds
inhibited PPO activity, the cis-form had higher inhibitory ability because it can bind more tightly to the active site
of the enzyme than its isomer.
Oms-Oliu et al. (2010) reviewed some recent advances in the maintenance of fresh-cut fruit quality with respect to
the use of chemical compounds, including plant natural antimicrobials and antioxidants, as well as calcium salts for
maintaining texture. They focused on the use of natural preservatives, which are of increasing interest because of the
toxicity and allergenicity of some traditional food preservatives, and on the difficulties in the application of these
substances to fresh-cut fruit without adversely affecting the sensory characteristics of the product. Edible coatings are
presented as an excellent way to carry additives since they have been shown to maintain high concentrations of
preservatives on the food surface, reducing the impact of such chemicals on overall consumer acceptability of fresh-
cut fruit.
C. Thermal Processing
Heat treatment is the most widely used method for stabilizing foods because of its capacity to destroy microor-
ganisms and to inactivate enzymes. Blanching is the most common method used to inactivate vegetable enzymes
(Marshall et al., 2000). It causes denaturation and therefore inactivation of the enzymes but also causes destruction of
thermosensitive nutrients, and it is rarely used for soft fruits (Lado and Yousef, 2002) because it results in losses in
vitamins, flavor, color, texture, carbohydrates, and other water-soluble components.
The inactivation of PPO as well as other spoilage enzymes can be achieved by subjecting the food article to
high temperatures for an adequate length of time to denature the protein. In general, exposure of PPO to
temperatures of 70e90�C destroys their catalytic activity, but the time required for inactivation depends on the
product (Chutintrasri and Noomhorm, 2006). Fortea et al. (2009), studying thermal inactivation of grape PPO and
peroxidase, described that they showed similar thermostability, losing over 90% of relative activity after only
5 minutes of incubation at 78�C and 75�C, respectively. Khandelwal et al. (2010) estimated the concentrations of
polyphenol in different cultivars of four pulses commonly consumed in India and examined the effects of
domestic processing. They demonstrated that processing reduced the concentrations of phenolic compounds by
19e59%. Chutintrasri and Noomhorm (2006), studying thermal inactivation of pineapple PPO, described that the
enzyme activity reduced by approximately 60% after exposure to 40e60�C for 30 minutes. Denaturation
increased rapidly above 75�C. Thus, residual activity was about 7% after 5 minutes at 85�C and 1.2% after
5 minutes at 90�C. In another study, to determine the effect of heating conditions on enzymatic browning,
Krapfenbauer et al. (2006) evaluated heated apple juice from eight different varieties of apples at high
temperature (60e90�C) and short time (20e100 seconds) (HTST) combinations. The results showed that HTST
treatment at 80�C already inactivated PPO, whereas pectinesterase activity was reduced to half and could not be
inactivated completely, even at 90�C. The highest residual pectinesterase activity was found at 60�C. Heating at
70�C caused stable pectinesterase activity and even a slight increase for 50 and 100 second heating times. In
Victoria grape PPO (Rapeanu et al., 2006), complete inactivation was reported after 10 minutes at 70�C
(Figure 10.9). The activity of PPO from Castanea henryi nuts after incubation at 70�C for 30 minutes was 8%
(Xu et al., 2004).
FIGURE 10.9 Thermal stability of Victoria grape polyphenol oxidase extract. Residual activity was measured after a 10-minute treatment at
different temperatures. (Reprinted from Rapeanu et al., 2006). � 2006, with permission from Elsevier.
409IV. Control or Inhibition of Enzymatic Browning
The heat inactivation of enzymes in foods is not only dependent on time but is also affected by pH. The optimum
temperature for PPO varies considerably for different plant sources as well as among cultivars, as shown in Table
10.2, and with the substrate used in the assay.
D. High-Pressure Processing
High hydrostatic pressure (HHP) treatment of fruit and vegetable products offers a natural, environmentally friendly
alternative to pasteurization and the chance to produce food of high quality, greater safety, and increased shelf-life
(Welti-Chanes et al., 2005). HHP reduces microbial counts and inactivates enzymes (Bayındırlı et al., 2006). The
HHP treatment is expected to be less detrimental than thermal processes to low-molecular-mass food compounds
such as flavoring agents, pigments, and vitamins, as covalent bonds are not affected by pressure (Butz et al., 2003). In
cashew apple juice processed by HHP at 250 or 400MPa for 3, 5, and 7 minutes, there was no change in pH, acidity,
total soluble solids, ascorbic acid, or hydrolyzable polyphenol content. These data demonstrate that HHP can be used
in the food industry, generating products with higher nutritional quality (Queiroz et al., 2010). Perera et al. (2010),
studied cubes of two different varieties of apple, which were vacuum packed in barrier bags with 0e50% (v/v)
pineapple juice and subjected to HHP at 600MPa for 1e5 minutes (22�C). They observed no visible color changes
during 4 weeks of storage in these bags. However, they also noticed that the texture and the in-pack total color change
after 5 hours’ air exposure were significantly affected by the apple variety, HHP time, and percentage of pineapple
juice used. The combined treatment significantly reduced residual PPO activity, whereas pectinmethylesterase
activitywas not affected in either variety.
HHP can affect protein conformation and lead to protein denaturation, aggregation, or gelation, depending on the
protein system, the applied pressure, the temperature, and the duration of the pressure treatment. Protein denaturation
is associated with conformational changes and it can change the functionality of the enzyme by increasing or losing
biological activity or changing substrate specificity. The effectiveness of treatment depends on the type of enzyme,
pH, medium composition, temperature, time, and pressure level applied (Hendrickx et al., 1998).
For several PPOs, it has been reported that pressure-induced inactivation proceeds more rapidly at lower pH and
that it is influenced by the addition of salts, sugars, or chemical antibrowning effectors (Rapeanu et al., 2005). It is
known that PPO is more resistant to pressure than thermal treatment, when compared to other enzymes (Y.-S. Kim
et al., 2001). In general, HHP is more effective at pressures above 600MPa (Figure 10.10), but the combination of
these methods increases the effectiveness of inactivation. The best results from studies aiming to decrease PPO
activity in banana purée (Palou et al., 1999), litchi (Phunchaisri and Apichartsrangkoon, 2005), and carrot juice (Y.-S.
Kim et al., 2001) were obtained at pressures higher than 400MPa combined with mild heat (50�C). In contrast, low
pressure (up to 400MPa) induced PPO activation in pear (200e400MPa, 25�C, 10 minutes) (Asaka and Hayashi,
1991) and apple juice (100MPa, 1 minute) (Anese et al., 1995).
HHP can be used to create new products (new texture or new taste) or to obtain analogue products with minimal
effects on flavor, color, and nutritional value, and without any thermal degradation (Messens et al., 1997). Pressure
can also influence biochemical reactions by reducing molecular spacing and increasing interchain reactions
(Marshall et al., 2000).
E. Gamma Irradiation
Irradiation is a physical treatment involving direct exposure to electrons or electromagnetic rays, for food preser-
vation and improvement of safety and quality. For these reasons, it can be used to extend the shelf-life of fruits and
vegetables. Radiation inactivates microorganisms, guaranteeing disinfection and delaying the ripening process and
senescence (Lacroix and Ouattara, 2000). In addition, retention of ascorbic acid and synthesis of polyphenols during
storage of irradiated food have been reported (Moussaid et al., 2000; Zhang et al., 2006).
Low-dose g-irradiation is commonly applied to fruit and vegetable products to extend shelf-life. Prakash et al.
(2000) noticed that treatment with a dose of 0.35 kGy decreased 1.5 and 1 log of total aerobic microorganisms and
yeasts and molds in cut Romaine lettuce, respectively, and that this dose did not adversely affect sensory attributes,
such as visual quality or off-flavor development. Latorre et al. (2010) observed the effect of low doses of g-irradiation
(1 or 2 kGy) on PPO activity of fresh-cut red beetroot and concluded that it produced biochemical changes in cellular
contents as well as in the cell wall constitutive networks which could not necessarily be sensed by consumers. They
also mentioned changes involving an increase in the antioxidant capacity of red beetroot tissue, showing that the
studied doses could be used in the frame of a combined technique for red beet processing. D. Kim et al. (2007) related
FIGURE 10.10 Effect of high hydrostatic pressure treatments and initial pH on residual polyphenol oxidase activity of avocado purée.
(Reprinted from Lopez-Malo et al., 1998). � 1998, with permission from Elsevier.
410 Chapter | 10 Enzymatic Browning
that total aerobic bacteria in fresh kale juice, prepared by a general kitchen process, were detected in the range of
106 cfu/ml, and about 102 cfu/ml of the bacteria survived in the juice in spite of g-irradiation treatment with a dose of
5 kGy. In the inoculation test, the growth of the surviving B. megaterium and E. acetylicum in the 3e5 kGy-irradiated
kale juice retarded and/or decreased significantly during a 3 day postirradiation storage period. However, there were
no significant differences in the residual PPO activity and browning index between the non-irradiated control and the
irradiated kale juice during the postirradiation period. Lu et al. (2005) showed a decrease in PPO activity of 73% in
fresh-cut celery treated with 1.0 kGy (0.5 kGy/hour) after 3 days under refrigeration (4�C). In 9 days, the enzyme
showed an activity around 25% lower than the control sample.
The use of irradiation in combination with other methods, or chemical antibrowning inhibitors, can result in
products that are microbiologically safe and of high quality (X. Fan et al., 2005).
F. Pulsed Electric Field
Pulsed electric field (PEF) is a non-thermal food preservation technology, used as an alternative to other conventional
techniques, which consists of introducing the food into a chamber containing two electrodes that apply high-voltage
pulses (20e80 kV) for a short time (microseconds).
In the past few years, several studies have demonstrated the ability of intense treatments to obtain safe and shelf-
stable liquid foods. Novel applications such as the improvement of mass transfer processes or the generation of
bioactive compounds using moderate field strengths are currently under development (Soliva-Fortuny et al., 2009).
This emerging food processing technology is being investigated owing to its ability to inactivate undesirable
microorganisms and enzymes, with limited increase in food temperature. As a result, more stable foods with fewer
changes in composition, physicochemical properties, and sensory attributes can be obtained (Martı́n-Belloso and
Elez-Martı́nez, 2005). The PEF applied to the food causes electroporation, that is, the irreversible loss of cell
membrane functionality, leading to inactivation of the microbial cell (Zhong et al., 2005; Cserhalmi et al., 2006), and
it also induces changes in the secondary structure of some enzymes. After PEF treatment, the loss of the relative a-
helix fractions of PPO and peroxidase were calculated and these results showed that PPO was more susceptible to the
treatment than peroxidase (Zhong et al., 2007).
411IV. Control or Inhibition of Enzymatic Browning
There are many studies in the literature focusing on the effect of PEF in the inactivation of microorganisms
(Garcı́a et al., 2003, 2005; Evrendilek et al., 2004; Li and Zhang, 2004), but few about the effect of this technology on
PPO activity (Giner et al., 2001, 2002; Zhong et al., 2007). The inactivation of commercial PPO depends on the
electric field strength and treatment time. The greatest reduction in PPO activity was 76.2% at 25 kV/minute for
744 ms (Zhong et al., 2007). Giner et al. (2001, 2002) reported a decrease of 97% on the PPO activity in apple extract
at 24.6 kV/cm for 6000 ms, 72% in pear at 22.3 kV/cm for the same treatment time, and 70% in peach PPO at 24.3
kV/cm for 5000 ms. Riener et al. (2008) observed a decrease of 71% in PPO activity using a combination of pre-
heating to 50�C and a PEF treatment time of 100 ms at 40 kV/cm in freshly prepared apple juice. This level of
inactivation was significantly higher (p< 0.05) than that recorded in juice processed by conventional mild
pasteurization, where the activity of PPO decreased by 46%.
According to Yang et al. (2004) the sensitivity to PEF treatment varies from enzyme to enzyme. The sequence of
sensitivity to PEF of five enzymes tested was: pepsin > PPO > peroxidase > chymotrypsin and lysozyme. The
inactivation effect of PEF on enzymes was affected by electric field strength, electrical conductivity, and pH.
G. Other Technologies
Although most studies have focused on HHP, irradiation, and PEF treatments, other emerging techniques may hold
promise for food technology.
Ohmic heating is defined as a process in which electric currents are passed through foods in order to heat them.
The heating methodaffects the temperature distribution inside a food, and directly modifies the timeetemperature
relationship for enzyme deactivation. Ohmic heating is distinguished from other electrical heating methods by the
presence of electrodes contacting the foods, the frequency, and the waveform of the electric field imposed between
the electrodes. Icier et al. (2008) evaluated fresh grape juice ohmically heated at different voltage gradients (20, 30,
and 40 V/cm) from 20�C to 60, 70, 80, or 90�C and the activity of PPO. The critical inactivation temperatures
were found to be 60�C or lower for 40 V/cm, and 70�C for 20 and 30 V/cm. The activation energy of PPO inactivation
for the temperature range 70e90�C was 83.5 kJ/mol. According to Castro et al. (2004), the inactivation kinetics of
PPO were first order for conventional and ohmic heating treatments. These authors demonstrated that, using this
technology, the time needed for enzyme inactivation was reduced. Icier et al. (2006) showed that ohmic heating can
also be used for blanching food products. After treatment by ohmic blanching, pea purée peroxidase was inactivated
in a shorter processing time than by conventional water blanching.
Supercritical carbon dioxide is a non-thermal technology in which a pressurization step ensures that the applied
gas penetrates the microbial cells and subsequent explosive decompression results in rapid gas expansion within the
cells, physically destroying them (Corwin and Shellhammer, 2002). Supercritical carbon dioxide has an effect on
enzyme inactivation due to conformational changes caused by gas in the secondary and tertiary structure (Gui et al.,
2007). According to a study conducted by Gui et al. (2007), cloudy apple juice exposed to supercritical carbon
dioxide at 30 MPa and 55�C for 60 minutes presented a reduction in PPO activity of over 60%, compared to a 27.9%
reduction obtained under atmospheric conditions at 55�C, indicating that the combined effects of pressure,
temperature, and time occurred after carbon dioxide treatment. Corwin and Shellhammer (2002) demonstrated the
inhibition of PPO activity, with a residual activity of 57.6% (500MPa, 3 minutes at 50�C), even when the carbon
dioxide was added before HHP. In this study, addition of carbon dioxide decreased the PPO activity significantly at all
pressures (0, 500, and 800MPa) and temperatures (25 and 50�C) beyond the effects of pressure alone. Niu et al.
(2010) evaluated the qualities of cloudy apple juices from apple slices treated with high pressure carbon dioxide and
mild heat, and observed that PPO was completely inactivated and its minimal residual activity at 65�C was 38.6%.
Ultrasound is a non-thermal technology; it causes enzyme inactivation by cell lysis using vibration energy, which
produces cavitation bubbles and temporarily generates spots of extremely high pressure and temperature when
imploded (Morris et al., 2007). Ultrasonication has been found to be more effective in inhibiting enzyme activity
when combined with other processes, such as high pressure and/or heat, contrary to the minimal inhibitory effects of
individual application. The effects of ultrasound and ascorbic acid on changes in activity of PPO and peroxidase of
fresh-cut apple during storage were investigated by Jang and Moon (2011) and Jang et al. (2009). They found that the
combined treatment inactivated monophenolase, diphenolase, and peroxidase, whereas individual treatment with
ultrasound or ascorbic acid had an inverse and limited inhibitory effect on the enzymes. This investigation revealed
that simultaneous treatment with ultrasound and ascorbic acid had synergistic inhibitory effects on several enzymes
related to enzymatic browning.
412 Chapter | 10 Enzymatic Browning
Blanching in hot water or steam has been widely used for thermal inactivation of undesirable enzymes, including
PPO. Because thermal treatments are also responsible for undesirable changes in tissue texture, several other methods
have been tried to inhibit PPO activity and avoid color changes in fruits (Lamikanra, 2002). Microwave heating is an
alternative method for liquid food pasteurization and, compared to the conventional method, microwaves can heat
products internally, and have greater penetration depth and faster heating rates that could potentially improve the
retention of thermolabile constituents in food (Heddleson and Doores, 1994; Deng et al., 2003). According to
Cañumir et al. (2002), microwave energy induces thermal effects in microorganisms and enzymes similar to those of
conventional heating mechanisms. Matsui et al. (2007) submitted solutions simulating the chemical constituents of
coconut water to a batch process in a microwave oven, and observed that PPO activity in water and in sugar solution
reduced after treatment. In salt solution, the stability of PPO was significantly affected and the contact between salt
and enzyme promoted a drastic reduction in the initial activity. The authors found that, at temperatures above 90�C,
the combined effects of salts and microwave energy reduced enzymatic activity to undetectable levels. However, at
90�C, the inactivation effect could be due to temperature alone. In a further study, Matsui et al. (2008) showed that
thermal inactivation of PPO during microwave processing of green coconut water was significantly faster than
conventional processes reported in the literature.
Short-wave ultraviolet light (UV-C) radiation is an alternative and inexpensive method to reduce the number of
microorganisms on the surface of fresh and cut fruits and vegetables (Yaun et al., 2004; Fonseca and Rushing, 2006;
Shama, 2006). The potential of UV-C light for commercial use in minimal processing of fruits depends on its ability
to contribute to food safety without causing undesirable quality changes. Few studies have been conducted on the
influence of UV-C on produce quality (color, texture, taste, and aroma) during storage. The reported effects are quite
diverse depending on the type of produce and the dosages applied (Shama, 2006). Although it has been claimed that
UV-C treatment does not produce undesirable by-products that could change taste, odor, and color, skin discoloration
in tomatoes, browning of calyxes in strawberries, and increasing susceptibility to brown rot in peaches have been
reported (Shama and Alderson, 2005). Erkan et al. (2001) found that high doses of UV-C caused a slight reddish-
brown discoloration on the surface of zucchini squash. Gómez et al. (2010) examined the effect of UV-C irradiation at
different doses, associated with some pretreatments (hot water blanching, dipping into a solution containing ascorbic
acid and calcium chloride) in order to minimize browning on the surface color of fresh-cut apple disks. They found
that the color and compression parameters were dependent on UV-C dose, storage time, and type of pretreatment.
Samples exposed to only UV-C light turned darker and less green compared to fresh-cut apple slices or to samples on
day 0, and this effect was more pronounced at the greatest UV-C dose. Light microscopic images showed the
breakage of cellular membranes in UV-C-treated samples, which may explain the increase in browning of irradiated
apples.
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	10 - Enzymatic Browning
	I. Introduction
	A. Historical Aspects of Polyphenol Oxidase
	II. Characteristics of Polyphenol Oxidase
	A. Structure and Sequence
	B. Mechanism of Reaction
	C. Biological Significance of Polyphenol Oxidase in Plants
	D. Phenolic Compounds in Food Material
	1. Simple Phenols
	2. Phenolic Acids
	3. Cinnamic Acid Derivatives
	4. Flavonoids
	E. Laccase
	F. Specificity of Polyphenol Oxidase
	III. Polyphenol Oxidase in Foods and Food Processing
	A. Role in Tea Fermentation
	B. Shrimps and Crustaceans
	IV. Control or Inhibition of Enzymatic Browning
	A. Exclusion of Oxygen
	B. Chemical Inhibitors of Polyphenol Oxidase
	C. Thermal Processing
	D. High-Pressure Processing
	E. Gamma Irradiation
	F. Pulsed Electric Field
	G. Other Technologies
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